Sustained release formulations of insect repellent

ABSTRACT

A microsuspension system containing insect repellent is disclosed that includes lipospheres, that are solid, water-insoluble microparticles that have a layer of a phospholipid embedded on their surface. The core of the liposphere is a solid insect repellent, or an insect repellent dispersed in an solid vehicle, such as a wax. Insect repellents include compounds that repel insects, are insecticidal, or regulate or inhibit insect growth on humans and other animals and plants. Insect repellent lipospheres provide controlled release of insect repellents while minimizing absorption through the skin.

This is a continuation of U.S. Ser. No. 07/607,542 which is abandonedwhich in turn is a continuation-in-part of U.S. Ser. No. 07/435,546,entitled "Lipospheres for Controlled Delivery of Substances," filed onNov. 13, 1989, by Abraham J. Domb, now abandoned.

BACKGROUND OF THE INVENTION

This invention is in the area of controlling delivery devices for theadministration of insect repellents.

Insects such as mosquitos, flies and fleas are a significant factor inthe spread of many serious diseases of man and other animals. Examplesinclude malaria, encephalitis, and a variety of parasites. They are alsoa general nuisance to humans and other animals. Efforts to repel theseinsect pests, other than with physical means, are generally ineffectivefor more than a few hours. There is also a significant economic lossassociated with animals spending their efforts repelling insect ratherthan foraging.

Most means for repelling insects consist of applying an organic compoundsuch as N,N-diethyl-m-toluamide (DEET) to the skin in an organicsolvent. DEET is the most effective compound known to repel mosquitos,however, even its effectiveness is limited to a few hours. Moreover, thesolvent in which it is applied to the skin also facilitates passage ofthe DEET through the skin, leading to undesirable toxicity, as well asirritation of the skin and eyes.

A variety of other compounds have been used to repel or kill otherinsects, especially flies and fleas. Most flea repellents orinsecticides consist of a combination of active ingredients, many ofwhich are not stable over a prolonged period of time. Efforts to extendthe period of the effectiveness have not met with great success,although some microencapsulated forms are being marketed. These consisteither of polymer or protein microcapsules incorporating the activeagent to be released. The disadvantages of these reagents are that theyare expensive to manufacture, they contain polymer residuals, and theyare gritty and uncomfortable when applied to the skin.

It is therefore an object of the present invention to provide acomposition and method to release insect repellents over an extendedperiod of time, especially on the skin of a human or other animal.

It is a further object of the present invention to provide a compositionand method to release insect repellents with minimum absorption throughthe skin or release of irritating chemicals at the application site.

It is another object of the present invention to provide a composition,and method of use thereof, for release of insect repellents, that iseasy to prepare and stable for an extended period of time prior to useand in vivo.

SUMMARY OF THE INVENTION

Solid, water-insoluble lipospheres including a solid hydrophobic coreformed of an insect repellent, alone or in combination with a carrier,having a layer of a phospholipid embedded on the surface of the core,are disclosed for use in providing extended release of active agent.

Lipospheres can be prepared by: (1) forming a liquid solution orsuspension of the insect repellent by either melting the repellent, ordissolving or dispersing the repellent in a liquid vehicle to form aliquid repellent that solidified at room temperature or greater; (2)adding phospholipid and an aqueous solution to the liquid repellent toform a suspension; (3) mixing the suspension at a temperature above themelting temperature until a homogeneous fine dispersion is obtained; andthen (4) rapidly cooling the dispersion to below the melting temperatureof the liquid mixture containing the repellent. The lipospheres formedby this process have a diameter of greater than one micron coated with alayer of a phospholipid. The hydrophobic side of the phospholipid isembedded in the surface of the solid hydrophobic core and thehydrophilic side of the phospholipid interfaces with the aqueoussolution.

Lipospheres can be designed to release repellent over a period ofseveral hours to approximately four or five days, or longer, by varyingthe ratio of carrier to repellent in the core, by choice of carrier,concentration of formulation and amount of liposphere administered. Forapplication of an insect repellent such as DEET to human skin, it ispreferred to have release for between several hours and one day.

Examples demonstrate the sustained release of DEET over a period of dayswhen applied topically to human skin, as measured by its effectivenessin repelling mosquitos.

DETAILED DESCRIPTION OF THE INVENTION

A delivery system for insecticides and insect repellents is describedthat results in an extended period of release of active agent. As usedherein, "insect repellent" refers to compounds killing, repelling orpreventing an increase in insect growth or populations. The deliverysystem are the lipospheres described in co-pending U.S. Ser. No.07/435,546 entitled "Lipospheres for Controlling Delivery ofSubstances," filed on Nov. 13, 1989, by Abraham J. Domb.

The lipospheres are distinct from microdroplets, vesicles or liposomessince the lipospheres have solid inner cores at room temperature. Thelipospheres are distinct from microspheres of uniformly dispersedmaterial in homogenous polymer since they consist of at least twolayers, the inner solid particle and the outer layer of phospholipid.

The combination of solid inner core with phospholipid exterior confersseveral advantages on the lipospheres as compared with conventionalmicrospheres and microparticles, including high dispersibility in anaqueous medium, and a release rate for the entrapped substance that iscontrolled by the phospholipid coating and the carrier. There are alsomany advantages over the other dispersion based delivery systems.Lipospheres have increased stability as compared to emulsion baseddelivery systems, including vesicles and liposomes, and are moreeffectively dispersed than most suspension based systems. Further, thesubstance to be delivered does not have to be soluble in the vehiclesince it can be dispersed in the solid carrier. Lipospheres also have alower risk of reaction of substance to be delivered with the vehiclethan in emulsion systems because the vehicle is a solid inert material.Moreover, the release rate of the substance from the lipospheres can bemanipulated by altering either or both the inner solid vehicle or theouter phospholipid layer. Lipospheres are also easier to prepare thanvehicles such as lipospheres, and are inherently more stable. Stabilityhas become the major problem limiting the use of liposomes, both interms of shelf life and after administration to a patient. Liposomes andvesicles do not remain intact or available in vivo after injection formore than a few hours to a couple of days. Unlike many of thebiodegradable polymeric systems, the lipospheres not made withbiodegradable polymers are stable in aqueous solutions. As importantly,the cost of the reagents for making the lipospheres (food grade) issignificantly less than the cost of reagents for making liposomes, whichrequire very pure lipids. For example, food grade lecithin costs about$2 per pound (from Central Soya Co., Ft. Wayne, Ind.), as compared withliposome grade lecithin which costs about $500 per pound (Avanti PolarLipids Inc., Pelham, Ala.).

The lipospheres containing the insect repellents are administered to theenvironment or the skin of a human or other animal or repel or killinsects. The lipospheres are preferably administered to the environmentby spraying an aqueous dispersion of the lipospheres in the area whereinsect control is desired. The lipospheres are preferably administeredto a human or other animal by spraying an aqueous dispersion oflipospheres or by topically applying the lipospheres in an aqueoussolution, ointment, cream, or other appropriate carrier. The liposphereformulations are stored in aqueous buffer, freeze dried, or in aointment or cream base, in the freezer, refrigerator or roomtemperature, and are stable for an extending period of time.

PREPARATION OF THE LIPOSPHERES

The preparation and modification of lipospheres is described first withreference to the following general descriptions and then with referenceto the following non-limiting examples of the preparation andapplication of lipospheres.

Selection of the Solid Core of the Liposphere.

In the preferred embodiment, the liposphere contains a core that has amelting temperature equal to or greater than room temperature,approximately 25° C. For example, for preparation of lipospheres to beapplied to the skin, the core is prepared by choosing an insectrepellent that has a melting temperature of approximately 30° C., or bymixing the insect repellent in a carrier to produce a mixture having amelting point of approximately 30° C. The repellent, or repellent andcarrier, preferably has a melting point of less than 120° C. and isstable in the liquid form when mixed with hot aqueous media.

The carrier must be compatible with the insect repellent. Suitable solidcarriers are inert hydrophobic biocompatible materials with a meltingrange between 30° and 120° C. Examples are natural, regenerated, orsynthetic waxes such as beeswax and carnauba wax; cholesterol; fattyacid esters such as ethyl stearate, isopropyl myristate, and isopropylpalmitate; high molecular weight fatty alcohols such as cetostearylalcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol; solidhydrogenated castor and vegetable oils; hard and soft paraffins; hardfat such as tristearin; biodegradable polymers such as polycaprolactone,polyamides, polyanhydrides, polycarbonates, polyorthoesters, polyacticacids, and copolymers of lactic acid and glycolic acid; cellulosederivatives and mixtures thereof. These and other suitable materials areknown to those skilled in the art and most are commercially available,as demonstrated by the extensive list of suitable carrier materials inMartindale, The Extra Pharmacopoeia, The Pharmaceutical Press, 28thEdition pp 1063-1072 (1982).

The release rate of the repellent from the liposphere is dependent inpart upon the composition of the core, as well as the outer phospholipidlayer, and can be altered by varying the compositions appropriately.

It is often desirable to deliver a water soluble repellent to a targetedarea, or to control the release of a water soluble substance. Since theinner core of the liposphere is hydrophobic, it is necessary to decreasethe water solubility of the repellent before liposphere preparation.Methods to decrease water solubility include using a water insolublesalt or base, complex, or insoluble precursor form of the repellent;preincorporating the repellent into hydrophobic microparticles that canbe used as repellent particles; or preparing an aqueous medium that therepellent is less soluble in, for example, by adjustment of pH or ionicstrength, or by adding salts or additives. If the repellent is renderedless water soluble by adjustment of pH or ionic strength, the resultinglipospheres can be isolated by filtration or centrifugation andreconstituted with an appropriate buffer solution prior to use.

Active materials can be preincorporated into microparticles of ahydrophobic solid phase, such as tristearin (melting point 65° C. to 72°C.), that can then be incorporated into lipospheres with a vehiclehaving a lower melting point, such as ethyl stearate (melting point 35°C.), to avoid melting the tristearin particles containing the activematerial. In this form, the tristearin-active material particles are thehydrophobic "repellent" which is dispersed in the ethyl stearateliposphere. The formulations can then be freeze dried with standardtechniques and reconstituted prior to use. Selection of the PhospholipicCoating.

The solid core of the liposphere is coated with one or morephospholipids that are embedded into the surface of the solid coreduring manufacture. Mixtures of two or more phospholipids can be used tovary the surface properties and reactivity of the liposphere. In someembodiments the phospholipid may be selected to enhance adhesion of thelipospheres to the surfaces to which they are applied.

Pholpholipid

A phospholipid is a phosphorylated diacylglyceride molecule or itsderivative. The parent structure is diacylglycerol phosphate, orphosphatidic acid. Phosphatidyl choline (lecithin) is the choline esterof phosphorylated diacylglyceride. Synthetic lecithin are available withacyl chain lengths ranging from 4 to 19 carbons. The preferred lecithinsfor biological applications are those with alkyl chain lengths in thebiological range (10 to 18 carbons). Naturally occurring lecithin can beobtained from a variety of sources such as egg, bovine heart, or soybean. Unsaturated lecithins (dioleoyl; dilinoleoyl; alpha-palmitoyl,beta oleoyl; alpha palmitoyl, beta linoleoyl; and alpha oleoyl, betapalmitoyl), dianachidonyl lecithin (highly unsaturated and aprostaglandin precursor), and alpha palmito beta myristoyl lecithin arealso available.

A molecule somewhat structurally related to phosphatidic acid,sphingomyelin, is also suitable for use in the coating of lipospheres.

Certain phospholipids, such as phosphatidic acid, phosphatidyl serine,phosphatidyl inositol, cardiolipin (diphosphatidyl glycerol), andphosphatidyl glycerol, can react with calcium, causing aggregation orthe binding of lipospheres to cell membranes. These reactions can beminimized by combining these phospholipids with non-calcium bindingphospholipids such as phosphatidyl choline. Phosphatidic acid can beisolated from egg or prepared synthetically (dimyristolyl, dipalmitoyland distearoyl derivatives are available from Calbiochem). Bovinephosphatidyl serine is also available commercially (Sigma Chemical Co.,St. Louis, Mo.). Phosphatidyl inositol can be isolated from plant orbovine sources. Cardiolipin can be purified from bovine or bacterialsources. Phosphatidyl glycerol can also be purified from bacterialsources or prepared synthetically.

Phosphatidyl ethanolamine in the pure state self-aggregates in acalcium-independent fashion, and is believed to have strong tendenciesto aggregate with cell membranes. It should therefore be used incombination with non-aggregating phospholipids. Phosphatidylethanolamine is commercially available, isolated from egg, bacteria,bovine, or plasmalogen or as the synthetic dioctadecanoyl, dioleoyl,dihexadecyl, dilauryl, dimyristolyl and dipalmitoyl derivatives.

For cost efficiency, non-injectable food grade lecithins can be used toformulate the lipospheres, such as Centrolex™ or Actiflo™ 70SB, whichare lecithins extracted from soya beans manufactured by Central Soya,Ft. Wayne, Ind.

Steroids

Steroids such as cholesterol (a natural constituent of membranes),estrogens, (such as estriol, estrone, estradiol and diethylstilbestrol),and androgens (such as androstenedione and testosterone) cannot functionalone as the liposphere coating but may be incorporated into thephospholipic surface coating, as well as serve as the core material.

Amphiphiles

Amphiphiles can be added to the phospholipic coating to alter thesurface charge on the liposphere. Examples of amphiphiles that produce apositive charge on the coating are protonated along chain alkyl aminessuch as stearylamine or the corresponding secondary, tertiary orquaternary substituted amines. Examples of amphiphiles that produce anegative charge are arachidonic acid and other fatty acids.

Surfactants

The phospholipids can be substituted in part with surfactants such asTween.sup.™ (a hydrophilic class of surfactants), Span™ (a hydrophobicclass of surfactants), and polyethylene glycol surfactants. Selection ofRepellent.

A number of biologically active agents for control of insects are knownand commercially available which are suitable for delivery inlipospheres. The types of insects that can be targeted for controlinclude flying insects such as mosquitos, flies, wasps, yellow jackets,hornets and bees, ants, roaches, lice, fleas, and scabies, as well asmany insects that eat or lay their eggs or plants.

A preferred compound for repelling mosquitos and flies is DEET(N,N-diethyl-m-toluamide). A preferred compound for inhibiting insectmaturation and reproduction is the insect growth regulator(S)-methoprene [isopropyl(2E,4E,7S)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate], marketed asPrecor™ by Sandox Ltd. Preferred compounds for killing fleas arepyrethrins, piperonyl butoxide, N-octyl bicycloheptene dicarboximide,and 2,3:4,5-Bis (2-butylene) tetrahydro-2-furaldehyde. Other compoundsthat are marketed for insect control include dimethyl phthalate (DMP),1,3-ethyl hexanediol (EHD),1-(3-cyclohexan-1-ylcarbonyl)-2-methylpiperidine (CYM),1-(3-cyclohexan-1-ylcarbonyl)-2-piperdine (CYP), and phosphorothioicacid O,O-diethyl O-[6-methyl-2-(I-methylethyl)-4-pyrimidinyl]ester,marketed as Diazinon™ by Ortho.

Representative agents to repel insects from plants include plants oils(neem, citronella, eucalyptus, marcosa, turpentine); amines(2-(2-methylamino-ethyl)-pyridine, 3-(methylaminomethyl)-pyridine,cadaverine, 1,8-diaminooctane, spermine, polyethyl eneimine); pesticides(cypermethrin, permethrin, carbofuran, chlormequat, carbendazim,benomyl, fentinhydroxide); carboxylic acids (n-decanoic and dodecanoic),and natural products such as polydogial and azadirctin.

These insect repellents/insecticides/insect growth regulators can becombined with other agents for control of pests, such as rodenticides,or with agents that attract insects, such as pheremones or flavorings,to increase the effectiveness of insecticides or growth regulators. Theycan be formulated for topical application, for application by spraying,or by application as an aerosol.

These compounds can be combined with other agents such as fertilizers,herbicides (such as atrazine,1-chloro-4-ethylamino-6-isopropylamino-s-triazine) and fungicides foruse in treating or killing plants, topically or systemically. They canalso be combined with agents that provide a pleasant smell (such asbenzyl acetate, citronellol, rhodinol, phenylethyl alcohol, and bergametoil) or prevent odor or drying of the skin and hair (such asantiperspirants and deodorants and lotions), for topical application tothe skin.

The effective concentration is determined empirically by comparingeffectiveness of formulations containing different quantities of activeagents. A preferred concentration of lipospheres containing DEET isbetween 1 and 50% by weight. Melt preparation of Lipospheres.

In the preferred embodiment, lipospheres are prepared by: (1) meltingthe repellent, or dissolving or dispersing the repellent in a liquidvehicle to form a liquid repellent which can be solidified by decreasingtemperature; (2) adding phospholipid along with an aqueous medium to theliquid repellent at a temperature higher than the melting temperature ofthe liquid repellent to form a suspension of the repellent; (3) mixingthe suspension at a temperature above the melting temperature of theliquid repellent until a homogeneous fine preparation is obtained; andthen (4) rapidly cooling the preparation to below the meltingtemperature to solidify the liquid core.

Suitable methods of mixing the suspension include mechanical shaking orstirring, fine mixing using homogenizing and sonication.

Solvent Preparation of Lipospheres.

Alternatively, lipospheres can be prepared by solvent processing. Thismethod of preparation of lipospheres involves the use of a solvents. Therepellent, carrier, and phospholipid are dissolved or mixed in anorganic solvent. The solvent is then evaporated, and the resulting solidis mixed with an appropriate amount of buffer and mixing continued untila homogeneous mixture is obtained. The temperature is then reduced to10° C. with continuation mixing for approximately five minutes to form amilky suspension of lipospheres.

In an aqueous solution, the lipospheres form a uniform fine dispersionof microspheres coated with a layer of a phospholipid, with thehydrophobic side of the phospholipid embedded in the outermost layer ofthe solid hydrophobic core and the hydrophilic side at the aqueousinterface. The particle size, particle distribution, and phospholipidcoating can be altered by varying the concentration and properties ofthe solid vehicle, the lipid, and the mixing method.

The method of preparation of lipospheres, and resulting lipospherescontaining repellent, described herein is simple and is characterized byhigh loading, reproducibility, versatility, and stability. The methodand compositions are further illustrated by the following non-limitingexamples.

Lipospheres encapsulating DEET were prepared. The liposphereformulations are superior to the formulations now being marketed inseveral respects. For example, the marketed preparations provide relieffor only few hours. Moreover, the concentration of DEET is thoseformulations is extremely high, about 75 percent. This is particularlyharmful because DEET is partially absorbed through skin. There have beenseveral reports of adverse systemic effects associated with the use ofDEET. The formulations described herein provide extended relief usinglow percent incorporation of DEET.

EXAMPLE 1 Preparation of Tristearin liposphere sustained releaseformulations of DEET.

500 mg of DEET was mixed with 1.0 gm of tristearin in a scintillationvial. The scintillation vial precoated with 500 mg of phosphatidylcholine (PCS). The scintillation vial was heated to 60° C. to obtain auniform solution of DEET and tristearin. 10 ml of 0.1M phosphate buffer,pH 7.4 was added to the vial and the contents were mixed by vortexing.After obtaining a uniform dispersion, mixing was continued withintermitted cooling in a dry ice/acetone bath. The resulting formulationwas a uniform, smooth textured paste.

Other formulations were prepared by varying the ratio of DEET,tristearin and PCS, as shown in Table 1.

EXAMPLE 2 Preparation of Polycaprolactone liposphere sustained releaseformulations of DEET.

500 mg of DEET was mixed with 1.0 gm of polycaprolactone in ascintillation vial. The scintillation vial was precoated with 500 mg ofphosphatidyl choline (PCS). the scintillation vial was heated to 60° C.to obtain a uniform solution of DEET and polycaprolactone. 10 ml of 0.1Mphosphate buffer, pH 7.4 was added to the vial and the contents weremixed using vortex. After obtaining a uniform dispersion, mixing wascontinued with intermitted cooling in a dry ice/acetone bath. Theresulting formulation was a smooth, uniform textured paste.

EXAMPLE 3 Preparation of DEET lipospheres using the solvent method.

To a round bottom flask containing 100 grams of glass beads (3 mm indiameter), 50 ml of chloroform was added. 1 gm of PCS, 1 gm oftristearin and 0.5 g of DEET was added to the flask and mixed thoroughlytill a clear solution was obtained. The chloroform was evaporated usinga rotoevaporator under reduced temperature at room temperature. Thetemperature was raised to 40° C. after 20 minutes, to ensure completeremoval of chloroform. A thin film of solids was obtained around thebottom flask and the glass beads. Ten milliliter of 0.9 % saline wasadded to the round bottom flask and the contents were mixed for 5 to 10minutes at room temperature. The temperature was then lowered to 10° C.by placing the flask in crushed ice and mixing was continued for anotherhalf hour.

The resulting lipospheres were spherical in shape, with an averageparticle size of between 8 and 15 microns.

EXAMPLE 4 Preparation and comparison of appearance of DEET liposphereshaving different liposphere formulations.

DEET formulations were prepared using the various compositions at aratio of 1:2:1 DEET, carried, and phosphatidyl choline, up to 10% inwater. The carriers used were: tripalmitin, beeswax, stearic acid,ethylstearate, and stearyl alcohol. The phosphatidyl cholines used were:lecithin from egg yolk and from soybean, and partially hydrogenatedphosphatidyl choline.

All formulations were milky in appearance with an average particle sizeof 10 to 30 microns.

EXAMPLE 5 Preparation of CCP repellent lipospheres.

Lipospheres containing 10% 1-(3-cyclohexen-1-ylcarbonyl)piperidine (CCP)were prepared by the method described in Example 1 or by the methoddescribed in Example 3. The lipospheres were either CCP:Tristearin: PCE1:2:1 or 1:1:1, by weight.

EXAMPLE 6 Demonstration of In vivo efficacy of tristearin andpolycaprolactone DEET lipospheres.

Efficacy Test:

In vivo efficacy tests were conducted on human volunteers who had giveninformed consent. The formulations prepared in examples 1 and 2 wereapplied to forearms at various doses, and subjects exposed to mosquitosat various time intervals. The experimental environment consisted of acylindrical chamber of fixed dimensions, equipped with 16×18 meshmosquito netting. The device contained between ten and twelve fastedmosquitoes having access to the skin through the mosquito netting. Theforearm was placed on the mosquito netting and the behavior of theinsects was observed. The time over which no mosquitos landed on theskin (100% repellency) was the index for determining the effectivenessof the formulation.

The results of the studies on the tristearin and polycaprolactone DEETlipospheres are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Effectiveness of DEET lipospheres.sup.a in repelling insects.                                            DEET        Effective-                             DEET   Core material                                                                            PCS      applied     ness                                   % (w/w)                                                                              % (w/w)    % (w/w)  mg/cm.sup.2                                                                          Area (hrs)                                  ______________________________________                                               Tristearin                                                             5      20         10        0.125 4    2                                      5      10          5        0.125 4    3.0                                    5      20         10        0.125 4    2.5                                    5       10*        5        0.125 4    3.5                                    0      20         10       0      4    0                                      10     10         10       0.25   4    3                                      10     30         10       0.25   4    4                                      15     30         10       0.50   4    4                                             Polycapro-                                                                    lactone                                                                5      10          5       0.50   19.63                                                                              2.5                                    5      20         10       0.50   19.63                                                                              2.2                                    5       10*        5                                                          0      20         10       0      19.63                                                                              0                                      15     30         10       0.75   19.63                                                                              5                                      ______________________________________                                         .sup.a 2.5 mg/cm.sup.2 of each formulation applied.                      

EXAMPLE 7 Preparation and efficacy of tristearin-PCE lipospherescontaining different DEET concentrations.

Eight formulations containing 5 and 10% W/W DEET (4 of eachconcentration) were prepared and tested for repellency activity. Allformulations contain tristearin as vehicle, egg yolk lecithin (PCE),DEET and phosphate buffer solution pH 7.4 added to 10 ml. Theformulations were prepared as described in Example 1 and tested asdescribed in Example 6 and 8. Table 2 summarizes the formulationcompositions and efficacy as determined according to example 6.

                  TABLE 2                                                         ______________________________________                                        Comparison of repellency of lipospheres                                       containing either 5% or 10% DEET.                                             Composition.sup.a (grams)                                                                         Period of Repellency                                      DEET     Tristearin  PCE    (hours)                                           ______________________________________                                        A    1.0.sup.b                                                                             1.5         0.5  5                                               B    1.0.sup.b                                                                             1           1    5                                               C    1.0.sup.b                                                                             2           0.5  >6                                              D    1.0.sup.b                                                                             3           1    4 to 5                                          E    0.5     0.75        0.25 2 to 3                                          F    0.5     0.5         0.5  2 to 3                                          G    0.5     1           0.25 2 to 3                                          H    0.5     1.5         0.5  2 to 3                                          ______________________________________                                         .sup.a One gram DEET is equal to 10.0% DEET.                                  .sup.b Testing and results described in examples 7 and 8.                

EXAMPLE 8 Comparison of the effectiveness of DEET lipospheres inrepelling two different species of mosquitos.

Method for testing repellency.

The repellent was applied to four locations on each arm. One ounce cupswith screen bottoms containing five avid (displaying host-seekingbehavior) mosquitoes, with Aedes aegypti or Anopheles stephensi, wereplaced on the treated portions of skin. Mosquitoes were also placed onan untreated section of the arm to serve as a control. After a tenminute period the cups were removed and the number of biting mosquitoes(evident by a blood meal) were counted. This procedure was repeated withpreviously unexposed mosquitoes every fifteen minutes in the first halfhour (with Aedes aegypti only) after application and on every half hourthereafter for a total of four hours after application. Both mosquitospecies tested: Aedes aegypti and Anopheles stephensi are extremelyaggressive biters in this test.

The formulations A, B, C, and D, described in Table 2, were applied at2.5 mg per cm² of skin surface. Formulations A (1.0 g DEET, 1.5 gtristearin, 0.5 g PCE to 10 ml with buffer) and C(1.0 g DEET, 2 gtristearin, 0.5 g PCE to 10 ml with buffer) were uniform in consistencyand applied easily, whereas B (1.0 g DEET, 1 g tristearin, 1 g PCE to 10ml with buffer) and D (1.0 g DEET, 3 g tristearin, 1 g PCE to 10 ml withbuffer) were more paste-like and proved difficult to spread.

                  TABLE 3                                                         ______________________________________                                        Comparison of effectiveness of DEET lipospheres against                       two species of mosquitos.                                                              Total number biting                                                  Time after                                                                             Aedes aegypti                                                                            (Anopheles stephensi)                                     application                                                                            Formulations                                                         (min)    A       B      C      D       Control                                ______________________________________                                        15    min    0       0    0      0       4                                    30           0 (0)   0 (0)                                                                              0 (0)  0 (0)   5 (0)                                60           0 (0)   0 (0)                                                                              0 (0)  0 (1)   5 (3)                                90           0 (0)   0 (0)                                                                              0 (0)  0 (1)   5 (2)                                120          0 (0)   0 (0)                                                                              0 (0)  0 (0)   5 (3)                                150          0 (1)   0 (0)                                                                              0 (0)  0 (0)   5 (5)                                180          0 (1)   0 (0)                                                                              0 (0)  0 (0)   4 (4)                                210          0 (1)   1 (1)                                                                              0 (0)  0 (1)   4 (2)                                240          0 (2)   0 (1)                                                                              0 (2)  0 (0)   0 (2)                                270          0 (2)   0 (2)                                                                              0 (2)  0 (1)   1 (5)                                ______________________________________                                    

Tested against Aedes Aegypti the formulations proved to be repellent,based on prevention of biting, for a minimum of 3.5 hours. At this pointthere was breakthrough biting on formulation B. Beyond this point intime no further biting occurred on treated surfaces, although biting onthe untreated surface dropped as well, presumably because this occurredat a time host-seeking behavior often ceases for Aedes aegypti.

Findings for Anopheles stephensi are not as clear cut. Biting on theuntreated surface is more erratic and there was early breakthroughbiting on some formulations, notably formulation D. By four hours therewas breakthrough biting on all formulations. The literature on mosquitorepellency has repeatedly reported limited repellency for DEET testedagainst Anopheline mosquitoes.

In conclusion, these formulations are repellent to Aedes aegypti for aminimum of four to six hours. It is likely that some of theseformulations are effective beyond this point. The fact that formulationC was effective against Anopheles stephensi for four hours, combinedwith the A. aegypti findings suggest that C is the most promisingrepellent tested at this time.

Modifications and variations of the present invention, lipospheredelivery systems for insect repellents, insect controls, andinsecticides, will be obvious to those skilled in the art from theforegoing detailed description. Such modifications and variations areintended to come within the scope of the appended claims.

I claim:
 1. A liposphere comprising:a core formed of a hydrophobicmaterial existing as a solid at a temperature of 25° C. and havinginsect repellent or insecticidal activity, and a phospholipid coatingsurrounding the core, wherein the hydrophobic ends of the phospholipidare embedded in the solid core and the hydrophilic ends of thephospholipid arm exposed on the surface of the liposphere, thecombination forming a spherical structure having an average particlediameter between 0.35 and 250 microns.
 2. The liposphere of claim 1,wherein the repellent or retardant activity is produced by a compoundincorporated into the hydrophobic core selected from the groupconsisting of insect repellents, insect growth regulators andinsecticides.
 3. The liposphere of claim 1, wherein the repellentcompound is selected from the group consisting ofN,N-diethyl-m-toluamide, (S)-methoprene, pyrethrins, piperonyl butoxide,N-octyl bicycloheptene dicarboximide, and 2,3:4,5-Bis (2-butylene)tetrahydro-2-furaldehyde, dimethyl phthalate, 1,3-ethyl hexanediol,1-(3-cyclohexan-1-ylcarbonyl)-2-methylpiperidine,1-(3-cyclohexan-1-ylcarbonyl)-2-piperidine, phosphorothioic acidO,O-diethyl O-[6-methyl-2-(I-methylethyl)-4-pyrimidinyl]ester, [plantsoils,] 2-(2-methylamino-ethyl)-pyridine, 3-(methylaminomethyl)-pyridine,cadaverine, 1,8-diaminooctane, spermine, polyethyl eneimine,cypermethrin, permethrin, carbofuran, chlormequat, carbendazim, benomyl,fentinhydroxide, n-decanoic and dodecanoic carboxylic acids, polydogialand azadirctin.
 4. The liposphere of claim 2 in a composition furthercomprising a compound selected from the group consisting of plantfertilizers, fungicides, rodenticides, herbicides, skin caresupplements, fragrances, antiperspirants and deodorants.
 5. Theliposphere of claim 4 wherein the solid core comprises the repellent ina vehicle for the repellent, which in combination with the repellentexists as a solid at 30° C.
 6. The liposphere of claim 5 wherein thevehicle is selected from the group consisting of natural, regeneratedand synthetic waxes, fatty acid esters, high molecular weight fattyalcohols, solid hydrogenated plant oils, solid triglyceride, andbiodegradable natural and synthetic polymers.
 7. The liposphere of claim1 wherein the repellent is N,N-diethyl-m-toluamide in a concentration ofbetween 1 and 50% by weight.
 8. The liposphere of claim 1 in a carrierfor topical administration to animals or to plants.
 9. A method forkilling or repelling insects comprising administering lipospheres formedofa core formed of a hydrophobic material existing as a solid at 25° C.having insect repellent or insecticidal activity, and a phospholipidcoating surrounding the core, wherein the hydrophobic ends of thephospholipid are embedded in the solid core and the hydrophilic ends ofthe phospholipid are exposed on the surface of the liposphere, thecombination forming a spherical structure having an average particlediameter between 0.35 and 250 microns.
 10. The method of claim 9 whereinthe repellent or insecticidal activity is produced by a compoundincorporated into the hydrophobic core selected from the groupconsisting of insect repellents, insect growth regulators andinsecticides.
 11. The method of claim 9 wherein the repellent compoundis selected from the group consisting of N,N-diethyl-m-toluamide,(S)-methoprene, pyrethrins, piperonyl butoxide, N-octyl bicycloheptenedicarboximide, and 2,3:4,5-Bis (2-butylene) tetrahydro-2-furaldehyde,phosphorothioic acid O,O-diethylO-[6-methyl-2-(I-methylethyl)-4-pyrimidinyl]ester, [plants oils,]2-(2-methyloamino-ethyl)-pyridine, 3-(methylaminomethyl)-pyridine,cadaverine, 1,8-diaminooctane, spermine, polyethyl eneimine,cypermethrin, permethrin, carbofuran, chlormequat, carbendazim, benomyl,fentinhydroxide, n-decanoic and dodecanoic carboxylic acids, polydogialand azadirctin.
 12. The method of claim 9 further comprising adding thelipospheres to a mixture comprising a compound selected from the groupconsisting of plant fertilizers, fungicides, rodenticides, herbicides,skin care supplements, fragrances, antiperspirants and deodorants. 13.The method of claim 9 wherein the solid core includes the insectrepellent and a vehicle for the repellent, which in combination with therepellent exists as a solid at 30° C.
 14. The method of claim 13 whereinthe vehicle is selected from the group consisting of natural,regenerated and synthetic waxes, fatty acid esters, high fatty alcohols,solid hydrogenated castor and vegetable oils, solid triglyceride, andbiodegradable
 15. The method of claim 9 for insect control furthercomprising preparing a dispersion of the lipospheres in an aqueoussolution and spraying the dispersion onto animals or plants.
 16. Themethod of claim 9 for insect control in an area further comprisingdispersing the lipospheres throughout the area where insect populationsare to be controlled.
 17. The method of claim 9 for repelling insectsfurther comprising dispersing the lipospheres in a carrier for topicalapplication to animals or plants.
 18. The method of claim 17 furthercomprising spreading the lipospheres on the skin of the animals to whichthe lipospheres are applied.
 19. The method of claim 9 furthercomprising preparing lipospheres by forming a non-aqueous liquid of thehydrophobic core material.adding phospholipid and an aqueous solution tothe liquid core material, and mixing the liquid core material andphospholipid until a suspension of lipospheres is formed.
 20. The methodof claim 19 further comprising sonicating and cooling the liquid corematerial and phospholipid after mixing to form the solid core.
 21. Themethod of claim 19 wherein the liquid core material and phospholipid areheated during mixing to a temperature equal to or greater than themelting temperature of the core material.
 22. The method of claim 19where the liquid core material includes an organic solvent, furthercomprising evaporating the solvent while mixing the core material andphospholipid.
 23. The method of claim 22 further comprising adding anaqueous buffer to the evaporated core material-phospholipid mixture andmixing until a suspension of lipospheres is formed.